Process for preparing conessine derivatives



United States Patent 0 3,539,449 PROCESS FOR PREPARING CONESSINE DERIVATIVES Arthur Friedrich Marx and Willem Frederik van der Waard, Delft, Netherlands, assignors to Koninklijke Nederlaudsche Gist-en Spiritusfabriek N.V., Delft, Netherlands, a corporation of the Netherlands No Drawing. Continuation-impart of application Ser. No. 568,896, July 29, 1966. This application Apr. 17, 1969, Ser. No. 817,161 Claims priority, application Netherlands, July 30, 1965, 6509936 Int. Cl. C07c 167/00 US. Cl. 195-51 1 Claim ABSTRACT OF THE DISCLOSURE Conessine derivatives of the class of 9a-hydroxy-cones sine, 12a-hydroxy-conessine, acid addition salts and quaternary ammonium compounds thereof are prepared by subjecting conessine to the action of enzymes of Botryodiplodia theobromae Pat, and recovering 9a-hydroxyconessine and l2a-hydroXy-conessine. These products can be, optionally, converted to acid addition salts and quaternary ammonium compounds.

REFERENCE TO A PRIOR APPLICATION This application is a continuation-in-part of application Ser. No. 568,896 filed on July 29, 1966, now US. Pat. 3,466,279, and entitled Conessine Derivatives and a division thereof.

In the Dutch patent application 280,926 a process is described according to which conessine is subjected to the action of micro-organisms; in this process conessine is converted into 3-oxo-4-conenine having the structural formula as shown below.

I CH3 N Torr CHa-l? O: l

C Ha

One of the micro-organisms which can be profitably used for this conversion is the fungus Stachybotrys parvispora. By increasing the assimilable carbohydrate content of the medium in which the said fungus was cultivated the primarily formed 3-oxo 4-conenine could be converted further almost quantitatively into llcc-hYdIOXY 4-conenine-3- one having the formula shown below.

This process is described in the Dutch patent application 6402112.

In the Dutch patent application 6405471 a process is described in which conessine is converted into 7oc-, 75-,

3,539,449 Patented Nov. 10, 1970 and lla-hydroxyconessine having the structural formula shown below.

CHa

by the use of enzymes of suitable fungi from the genera Gloeosporium, Colletotrichum, and Myrothecium. Furthermore a purely chemical method for the preparation of 3-oXo-1,4-conadienine illustrated below.

by starting from conessine is described in the US. patent specification 2,910,470.

It is an object of the present invention to provide a process for preparing 9u-hydroxyconessine and 12a-hydroxyconessine as well as acid addition salts and quaternary ammonium derivatives thereof.

Other objects will become more apparent as the description thereof proceeds.

It was now found that 9a-hydroxyconessine and 12ahydroxyconessine having the respective formulae as shown below:

can be prepared by subjecting conessine to the action of enzymes of Botryodiplodia theobromae Pat, which are formed by cultivtaing the micro-organism in a Raulin- Thom medium. According to the invention the compounds obtained, may be converted into acid addition salts or mo11oor bis-quaternary compounds. The salts can also be used for the isolation and/or purification of the reaction product.

Botryodiploa'ia theobromae Pat. is the imperfect form of Physalospora rhodina (Berk. et Curt.) Cke., isolated from infested coconut pulp.

In the process according to the invention preferably a submerged culture of Botryodiplodia theobromae Pat. is made to act under aerobic conditions on the starting product. Shaking or stirring may be applied. The conessine is preferably added to the culture in the form of a solution of a salt.

When the conversion to 9a-hydroxyconessine and 12ahydroxyconessine is complete, which is checked preferably by means of chromatography, the final product is isolated from the culture, preferably by filtration and extraction. With the aid of known methods, e.g., by conversion into functional derivatives, crystallization, and/ or extraction, the final products can be obtained separately in a state ofpurity.

The compounds 9a-hydroxyconessine and 12a-hydroxyconessine obtained according to the invention have not been described before. The said substances are intermediates for the preparation of the salts and the quaternary ammonium compounds, which can be used as muscular relaxing agents.

The salts include the monoand di-acid-addition salts, particularly non-toxic pharmacologically acceptable acidaddition salts. Acids useful in preparing the addition salts comprise, among others, organic acids, such as oxalic, tartaric, citric, succinic, acetic, fumaric, lactic and maleic acid; and inorganic acids, such as nitric, sulphuric, phosphoric, boric and especially hydrohalic acids, e.g. hydrobromic and hydrochloric acid.

The quaternary ammonium derivatives include monoand bis-quaternary ammonium compounds. These compounds are prepared by reacting the corresponding nonquaternized compounds with a quaternizing agent.

Suitable quaternizing agents are the familiar esters of aliphatic and araliphatic alcohols derived from strong acids. Aliphatic and araliphatic esters of sulphuric acid, hydrohalic acids, such as hydrochloric acid, hydrobromic acid, or hydroiodic acid, may be mentioned as examples. As alcohols, of particular importance are the lower alkanols, lower alkenols, phenyl-loWer-alkanols and cycloalkyl-lower-alkanols. The quaternizing esters are preferably ethyl iodide, methyl iodide, ethyl bromide, methyl bromide, methyl sulphate, allyl bromide, benzyl bromide, cyclohexylmethyl bromide, etc.

The quaternizing reaction is carried out in the conventional way, e.g. by boiling 9a-hydroxyconessine or 12a-hydroxyconessine in a suitable solvent, such as acetonitrile, alcohols, mixtures of alcohols and water, benzene, or acetone with an alkyl or aralkyl ester of a strong acid.

The invention also relates to pharmaceutical composi tions comprising a minor amount of at least one quaternary ammonium compound of 9nt-hydroxyor 12ot-hydroxyconessine and a major amount of a pharmaceutical carrier. The pharmaceutical compositions can be prepared in a usual way. The quaternary ammonium compounds in question are preferably dissolved in a physiological salt solution, may or may not be placed in particular doses in ampoules under an inert gas, and may subsequently be sterilized in the conventional way. The compositions can be used for human as Well as veternary practice.

The following examples serve to illustrate the process according to the invention, but are not to be construed as limiting the invention. For example, it is possible to use other culture media as well.

EXAMPLE I A medium according to Raulin-Thom, which contains g. of glucose, 2.7 g. of tartaric acid, 2.7 g. of ammonium tartrate, 0.4 g. of secondary ammonium phosphate, 0.4 g. of potassium carbonate, 0.3 g. of magnesium carbonate, 0.7 g. of ammonium sulfate, 0.05 g. or zinc sulfate, and 0.05 g. of ferrous sulfate per litre of water, is brought to pH=5 with 30% potassium hydroxide solution and sterilized for 20 minutes at 120 C.

A 2-litre flask containing 500 cm. of this culture medium, is inoculated from a tube with Botryodiplodia theobromate Pat. and shaken for three days at 26 C. Subsequently 4.5 litres of this culture are transferred to a 1500 litre vessel containing 200 litres of sterilized main fermentation medium, consisting of 5 g. of glucose and S g. of corn steep liquor-calculated as dry matter-per litre, the pH of which has been brought to 6.8 with sodium hydroxide solution, and 100 cm. of anti-foaming oil. The culture is kept at a temperature of 26 C., aerated with 200 litres of sterile air per minute and stirred at a rate of 150 r.p.m. Under steril conditions, 24 hours after the inoculation of the main fermentation medium a solution of 50 g. of conessine in dilute sulfuric acid of pH=2.0 is added and the mixture is stirred and aerated at the same temperature for another 22 hours. The conversion is found to have taken place as to about 90%. At the end of the process the pH is 7.4-7.6.

The fermentation broth is acidified with sulfuric acid to pH=23 and filtered. The filtrate is rendered alkaline with sodium hydroxide solution to pH=10 and extracted three times with one third its volume of methyl isobutyl ketone. The extract is concentrated and extracted with acid. The acid aqueous layer, after being made alkaline, is extracted once more with methyl isobutyl ketone. The extract is evaporated. The yield of crude product is calculated on conessine.

89.2 g. of the crude product is dissolved in 1 litre of pyridine. After addition of 125 g. of succinic anhydride, the mixture is heated for 6 hours to 100 C. and then kept overnight at room temperature. By evaporation under reduced pressure the pyridine is removed as much as possible. The residue is taken up in the system as much anol, water, and methyl isobutyl ketone. After the pH have been brought to 9.5, 15 g. of precipitate is formed, which according to chromatographic analysis is found to consist substantially of 9a-hydroxyconessine At the said pH the 12u-hydroxyconessine hemisuccinate is reasonably stable and remains dissolved as a salt. From the remaining aqueous layer 9.0 g. of hydroxyconessine in a state of impurity can further be obtained by extraction with methyl isobutyl ketone.

Recrystallization from methyl isobutyl ketone yields 15.9 g. of 9a-hydroxyconessine with a melting point of 205.5208 C. and [a] =-39 (c.=1.01 in chloroform).

Elemental analysis.Calcd. (percent): C, 77.42; H, 10.75; N, 7.52. Found (percent): C, 77.34; H, 10.81; N, 7.43.

The NMR spectrum gives the following 5 values with respect to tetramethyl silane in deuterochloroform after extraction with heavy water:

1.083 ppm. for the three protons attached to C-atorn 19 1.025 ppm. for the doublet of the three protons attached to C-atom 21 5.41 ppm. for the proton attached to C-atom 6 3.05 and 1.98 ppm. for the two doublets of the two protons attached to C-atom 18.

The aqueous layer, from which 9a-hydroxyconessine has been extracted, is now rendered more strongly alkaline by addition of cm. of 11 N sodium hydroxide solution, in consequence of which 12u-hydroxyconessine hemL succinate slowly decomposes. Extractions with methyl isobutyl ketone then yield 54.7 g. of crude product. By fractional recrystallization from benzene a preparation of 12nt-hydroxyconessine with a melting point of 257-259 C. (20.15 g.) is then obtained. [u] =-{-39 (c.=1.09 in chloroform).

Elemental anaIysis.-Calcd. (percent): C, 77.42; H,

10.75; N, 7.52. Found (percent): C, 77.22; H, 10.81; N, 7.74.

The NMR spectrum is characterized by the following 6 values with respect to tetramethyl silane in deuterochloroform after extraction with heavy water:

0.925 ppm. for the three protons attached to C-atom 19 1.025 ppm. for the three protons attached to C-atom 21. A doublet occurs.

5.35 ppm. for the proton attached to C-atom 6 3.86 ppm. for the proton attached to C-atom 12 3.01 and 1.78 p.p.m. for the two doublets of the two protons attached to C-atom 18.

Separate experiments have been carried out to determine the position of the hydroxyl groups and the configuration of the C-atoms carrying the hydroxyl groups. They are briefly described below.

(a) 12a-hydroxyconessine, which according to the infra-red spectrum and the elemental analysis contains a hydroxyl group, can be acetylated and oxidized. The oxidation product, obtained by treatment of lZu-hYdIOXY- conessine with a solution of chromium trioxide in 90% acetic acid, is identical with 12-oxo-conessine, obtained by oxidation of holarrhenine, which is known to be lZB-hydroxyconessine. The melting point found is l30-l31 C. and the results of the elemental analysis are:

Calcd. (percent): H, 10.27; C, 77.84; N, 7.57. Found (percent): H, 10.30; C, 77.65; N, 7.52 [a] =+32 (c.=1.1 in ethanol).

The infra-red spectrum gives an absorption of a sixring ketone. Reduction of the oxidation product with lithium aluminium hydride gives both holarrhenine and 12m-hydroxyconessine again, to be separated by crystallization. The two products have been identified by means of the melting points, the mixed melting points, the infra-red spectra, and the chromatographic R values.

(b) It is not possible to acylate 9a-hydroxyconessine. The presence of a tertiary hydroxyl group is confirmed by the fact that 9a-hydroxyconessine cannot be oxidized. The NMR spectrum points to the 9a-hydroxy compound. According to Ziircher (Helv. Chim. Acta 46, (1963) 2054) a chemical shift of the protons attached to C-atom 19 of 0.142 ppm. with respect to corresponding protons in conessine takes place, so that a 6 value of 1.072 was to be expected for this group. The value found was 6:1.083 ppm. The 9,B-hydroxyl group would cause a shift of 0.083 p.p.m. of the protons, so that this possibility is not very likely. More certainty concerning the position of the hydroxyl group in 9a-hydroxyconessine has been obtained in the following way:

9a-hydroxyconessine and the known compound llthydroxyconessine are subjected separately to dehydration. 9a-hydroxyconessine is boiled with toluene sulfonic acid in toluene and llot-hYdIOXYCOIlCSSiIlC is kept tosyl chloride in pyridine at 50 0, upon which in the latter case the ester formed is decomposed with sodium acetate in glacial acetic acid.

In both cases dehydration then takes place. After purification and crystallization, the two reaction products are compared as to their melting point, which is found to be 98-100 C., their mixed melting point, NMR spectrum, and infra-red spectrum. In both cases the dehydration product is A -conessine illustrated above.

having a melting point of 172l72.5 C. and [a] =42 (c.=0.5 in chloroform) and 9a-hydroxy-3fi-dimethylaminoconanine, shown below:

having a melting point of 203-204 C. and [a] =+35 (c.=0.5 in chloroform) respectively are obtained. According to the literature (Fieser and Fieser, Steroids (1959), p. 271) upon catalytic reduction of A -steroids almost exclusively the 500 compound is formed. An equatorial substitute at C-atom 3 does not affect the course of the reduction.

The hydrogenated 5a compounds show a more polar behavior in thin-layer chromatography than the corresponding hydrogenated 55 compounds. (Coll. Czech. Chem. Comm. 28 (1963) 2932). In this case again the Sec compounds are formed.

The next step is the dehydration of 1la-hydroxy-3fidimethylaminoconanine via its tosyl ester with sodium acetate in glacial acetic acid to form 3,6-dimethyla1nino- A -conenine having a melting point of ll0110.5 C. and [a] =44 (c.=0.5 in chloroform).

Infra-red spectra and NMR spectra are in conformity with the structure. 3fl-dimethylamino-A -conenine can be completely converted with perphthalic acid at 0 C. into 3B-dimethylamino-9u-1let-epoxyconanine 3-N-oxide, as appears from the NMR spectrum. The product cannot be obtained in the crystalline form.

The epoxide can then be converted into 3B-dimethylamino-9u-hydroxyconanine by reduction with lithium aluminium hydride. Identification is effected by means of a mixed-melting point determination with hydrogenated 9a-hydroxyconessine, elemental analysis, and infra-red analysis. Melting point 20 2203 C.; [a] =34 (c.'=0.5 in chloroform).

EXAMPLE II Preparation of 9a-hydroxyconessine bis-methiodide A mixture of 400 mg. of 9ot-hydroxyconessine, 2 ml. of freshly distilled methyliodide and 20 ml. of methanol is refluxed for 1 hr. The reaction mixture is then concentrated and the crystallized product filtered off and dried. The crude product (406 mg.) is crystallized from methanol-acetone 1:1; v./v.) yielding 264 mg. of pure 9a-hydroxyconessine bis-methiodide; M.P. 305-306 C. LR. (KBr): 3463, 3017, 1025, 960, 912, 840 cmr Calcd. for C H I N O (656) (percent): C, 47.56; H, 7.01; I, 38.72; N, 4.27. Found (percent): C, 47.82; H, 7.33; I, 38.63; N, 4.06.

EXAMPLE III Preparation of IZa-hydroxyconessine bis-methiodide In the same way 12u-hydroxyconessine is transformed into IZa-hYdIOXYCOIlBSSiHC bis-methiodide. The pure product has a M.P. of 293-294 C. LR. (KBr): 3350, 3015, 1040, 950, 910, 840 cmr Calcd. for C H I N O (656) (percent): C, 47.56; H, 7.01; I, 38.72; N, 4.27. Found (percent): C, 47.79; H, 7.28; I, 38.67; N, 4.07.

We claim:

1. A process for preparing conessine derivatives selected from the group consisting of 9a-hydroxy-conessine, 12a-hydroXy-conessine, organic and inorganic acid addition salts thereof, and quaternary ammonium derivatives thereof derived from esters of strong mineral acids and alcohols selected from the group consisting of loweralkanols, lower-alkenols, phenyl-loWer-alkanols, and cycloalkyl-lower-alkanols, the process comprising the steps of subjecting conessine to the action of enzymes of Botryodiplodia theobromae Pat, and recovering said 9a-hydroxyconessine and l2a-hydroxyconessine, and optionally converting the same into derivatives selected from the group consisting of acid addition salts by reaction withacids, and quaternary ammonium compounds, by reaction with esters of strong inorganic acids with alcohols selected from the group consisting of lower-alkanols, lower-alkenols, phenyl-lower-alkonols and cycloalkyl-loWeI-alkanols.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, Primary Examiner U.S. Cl. X.R. 260239.5 

